【作者】 李艳；

【导师】 岳天孚；

【作者基本信息】 天津医科大学 ， 妇产科学， 2012， 博士

【摘要】 研究目的1.收集术中切除的新鲜上皮性卵巢癌组织,建立具有多种组织学类型的大样本裸鼠移植瘤模型；观察移植瘤生长特点,探讨临床病理因素与移植瘤成瘤率的关系,并对比观察裸鼠移植瘤组织与原人卵巢癌组织、细胞系移植瘤组织的形态学特征。为系统性研究卵巢癌的发病、生物学特性、基因表型、临床前药效学检测等方面提供较好的研究平台。2.应用裸鼠荷人卵巢癌移植瘤模型,观察泰素(紫杉醇)、卡铂和顺铂对移植瘤的疗效,结合临床用药效果进一步探讨裸鼠荷人卵巢癌移植瘤模型在药效学方面的应用价值。3.采用高通量方法检测卵巢癌第五代裸鼠移植瘤组织的基因突变,结合文献筛选卵巢癌相关基因,进一步探讨在卵巢癌进展过程中是否存在某些信号转导通路的异常,为研发新的抗肿瘤药物提供依据。研究方法1.无菌条件下收集术中冰冻证实为上皮性卵巢癌的瘤组织,6h内接种至裸鼠背部皮下(4点/只),3-5只/例,观察原代移植瘤的生长情况。移植瘤6m内继续生长并且肿瘤体积达到500mm3者为原代成瘤,随后再次传代。连续传代过程中移植瘤在接种60天内体积能达到800mm3并进行传代者为建模成功。从第三代开始测量裸鼠体重和肿瘤体积,绘制生长曲线。将原发瘤、第一代移植瘤、第五代移植瘤和人卵巢癌细胞系移植瘤组织进行HE染色并对比观察其结构特点。2.选取ID号为17、34、49和50号的第五代移植瘤模型,每例均随机分为4组给予不同处理：生理盐水、泰素(紫杉醇注射液)、卡铂和顺铂。在实验过程中,根据各组平均移植瘤体积绘制生长曲线,计算肿瘤相对增殖率(T/C×100%,T为给药组移植瘤体积的平均值,C为对照组移植瘤体积的平均值),观察药物的抑瘤效果,并与临床治疗效果相比较。3.随机选取12例第五代移植瘤冻存组织。选用Agilent SureSelect靶向序列富集捕获法制备样本文库,借助Illumina Solexa HiSeq2000测序平台进行测序,将测序结果与美国国立生物技术信息中心的参考基因组build37.3进行比对,确定突变位点,结合HMGD(human mutation gene database)专业版(profession version)和BIC(breast cancer information core)数据库所公布的基因突变信息确定检测到的突变是否为SNPs (Single Nucleotide Polymorphisms, SNPs,单核苷酸多态性)以及是否是新的突变。结果1.依据原代人卵巢癌组织裸鼠移植瘤接种成功标准,原代接种成瘤率为53.13%(34/64),再次传代成功率为94.12%(32/34)。人卵巢癌组织接种到裸鼠体内后到第一次传代时间间隔以2-3m为多,随后的鼠间传代时间间隔多数为3-8w,少数为2w左右。所有裸鼠皮下移植瘤模型均没有腹水形成,且移植瘤的转移能力较弱。只有少数肿瘤具有很强的转移能力,接种后很快出现肝和脾转移,但此种转移能力随传代次数的增加而减弱。与人卵巢癌细胞系移植瘤相比,卵巢癌组织移植瘤与人原发瘤组织结构更相似。2.不同FIGO分期的人卵巢癌裸鼠移植瘤模型的成瘤率无显著性差别(x2=3.022；P=0.388)；4例粘液腺癌成瘤率为零,其它不同组织学类型之间成瘤率亦无显著差异(x2=0.997；P=0.802)。Ⅲ/Ⅳ期浆液性腺癌较Ⅰ/Ⅱ期浆液性腺癌在裸鼠体内更易成瘤(P=0.018),早期和晚期非浆液性腺癌的成瘤率无显著差别。而对于Ⅰ/Ⅱ期卵巢癌来说,非浆液性腺癌(子宫内模样癌、透明细胞癌和低分化癌)则较浆液性腺癌更易成瘤(P=0.044)。3.各例泰素组、卡铂组和顺铂组相对于空白对照组的T/C值如下：ID17号分别为44.93%、27.1%、11.77%；ID34号分别为65.15%、57.81%、30.67%；ID49号分别为49.50%、28.97%、6.79%；ID50号分别为68.97%、16.64%、34.02%。三种药物对这4例人卵巢癌移植瘤均表现出显著的抑瘤效果(P值均<0.01),与临床患者初始化疗疗效相似。4.12例卵巢癌移植瘤组织均发生了多个基因的点突变,一部分突变基因编码蛋白为EGFR相关信号转导通路中的上下游分子,如EGFR、KRAS、PIK3CA,AKT1、 mTOR等。EGFR合并KRAS突变者5例、合并PIK3CA突变者3例、合并AKT1突变者2例、合并mTOR突变者2例。12例标本中具有BRCA1和BRCA2突变者分别为7例和12例,其中合并TP53突变者分别为6例和10例,合并PARP1突变者分别为5例和10例。BRCA1、 BRCA2和TP53基因以多位点突变多见,而EGFR、KRAS、AKT1、PIK3CA、mTOR、PTEN及PARP1基因常表现为单一位点突变。12例移植瘤组织中CTNNB1和BRAF基因型均为野生型。通过检索HMGD专业版和BIC数据库,证实下述点突变为致病突变：EGFR的p.R521K突变,PARP1的p.V762A和p.K940R突变,BRCAl的p.W353Stop、p.P871L、p.E1038G、p.K1183R、p.S1613G、p.R1751Stop突变,BRCA2的p.N372H、p.H523R、p.13412V、p.A2466V突变,TP53的p.R72P、p.R273C、p.R175H、p.C141Y突变,上述突变中除BRCA2的p.H523R和TP53的p.C141Y两个突变外,其余突变均已被证实为SNPs。结论1.本研究建立的34例第一代裸鼠荷人卵巢癌移植瘤模型为今后进一步深入研究卵巢癌创建了一个很好的研究平台,但移植瘤可出现某些生物学行为的改变如生长方式、转移能力等。2.顺铂、卡铂和泰素在实验周期内均对移植瘤有很好的抑瘤效果,与临床治疗初期疗效相似,因此,裸鼠移植瘤模型可作为新药研发和检测肿瘤对药物敏感性的有效工具。3.经多次传代后移植瘤组织保持了原人卵巢癌的组织形态学特点及基因突变特征。更多还原

【Abstract】 Objectives1. To establish a variety of primary ovarian cancer xenograft models in nude mice using the fresh tissues of epithelial ovarian carcinoma under sterile condition from the operationroom. To observe the growth characteristics of the xenogrfts and discuss the relationship between the clinical-pathological factors and tumor take rate. Compare the morphological character of the xenografts derived from ovarian cancers and cell lines with the original human tumors. It provides a research platform for systematic study of pathogenesis, biological characteristics, preclinical pharmacodynamics experiment, and gene phenotype of ovarian cancer.2. To test the efficacy of taisu(taxol), carboplation and cisplation on the human ovarian carcinoma xenografts. To evaluate the application value of the nude mice xenograft models in pharmacodynamics by comparing the efficacy with clinical outcome.3. To detect the genes alterations in fifth-generation xenografts by Next Generation Sequencing and select the related genes with ovarian cancer by searching the literature. It provides the basis for new anticancer drug.Methods1. The collected epithelial ovarian cancer tissues which were confirmed by frozen sections were inoculated subcutaneously in nude mice within6hours (4spots per mouse,3-5mice per case). The growth of the xenografts was observed. The definition of tumor take rate is that the xenograft can grow progressively after implantation and could be serially passaged within6month. Xenografts which were larger than800mm3within60days during the following passage were regarded as success. The weight of the mice and the volume of xenografts were measured from passage3and the growth curves were drawn. The pathological features of P1xenografts, P5xenografts, the corresponding human ovanrian cancer and xenografts originated from ovarian cancer cell lines were compared.2. The P5xenografts of ID17, ID34, ID49and ID50were randomly devided into4 groups (blank controller, taisu, carboplatin, and cisplatin), respectively. The efficacy of the above drugs were evaluated by T/C value. T and C were the mean volume of the treated and control groups respectively on a given day. The experimental result and clinical efficacy were compared.3. The genes alterations were detected using Angilent SureSelect target enrichment capture system and Illumina Solexa HiSeq2000sequencing paltform. A total of12P5xenografts were analyzed. The results were compared with NCBI reference genome build37.3. The results were analysed by retrievaling HMGD in version of profession and BIC database.Results1. Overall tumor take rate was53.13%(34/64). The take rate of xenografts in subsequent passages was94.12%(32/34). The delay from initial implantation to the first passage was2-3months for most of the xenografts, and the time between subsequent passages was3-8weeks. No abdominal fluid was observed in the tumor bearing mice. Metastatic spread was observed in only3cases. The xenografts originated from human were more similar with human ovarian cancer than xenografts originated from ovarian cancer cell lines.2. There were no significant difference in tumor take rate among different FIGO stages (χ2=3.022;P=0.388) and among different histological types except for mucious adenocarcinoma (χ2=0.997;P=0.802). The tumor take rate of mucious adeno-carcinoma was0. The tumor take rate of Stage Ⅲ./Ⅳ serous adenocarcinoma was higher than Stage Ⅰ/Ⅱ (P=0.018). The tumor take rate of non-serous adenocarcinoms including endometrioid carcinoma, clear cell carcinoma, and low differential cancer was higher than serous adenocarcinoma for Stage Ⅰ/Ⅱ (P=0.044).3. T/C value of ID17was44.93%,27.1%,11.77%in taisu, carboplatin, and cisplatin group, respectively. T/C value of ID34was65.15%,57.81%,30.67%in taisu, carboplatin, and cisplatin group, respectively. T/C value of ID49was49.50%,28.97%,6.79%in taisu, carboplatin, and cisplatin group, respectively. T/C value of ID50was68.97%,16.64%,34.02%in taisu, carboplatin, and cisplatin group, respectively. All the drugs had anticancer activity (P<0.01of all), which were similar with clinical initial treatments.4. There were many mutation genes in12xenografts. Partial of them were the key molecules in EGFR signal transduction pathway such as EGFR, KRAS, PIK3CA, AKT1, mTOR. EGFR with KRAS, PIK3CA, AKT1, mTOR mutation simultaneously were presented in5,3,2,2cases respectively. BRCA1and BRCA2mutation were observed in7and12xenogratfs respectively. There were6cases with BRCA1and TP53mutation and10tumors with BRCA2and TP53mutation. Similarly, the number of cases with BRCAl and PARP1mutation or with BRCA2and PARP1mutation were5and10respectively. Multisite mutation was detected in genes of BRCA1, BRCA2, and TP53. Single point mutation was identified in genes of EGFR, KRAS, AKT1, PIK3CA, mTOR, PTEN and PARP1. CTNNB1and BRAF were wild-type in all of12xenegrafts. Harmful mutations in the study include p.R521K of EGFR; p.V762A and p.K940R of PARP1; p.W353Stop, p.P871L, p.E1038G, p.K1183R, p.S1613G and p.R1751Stop of BRCA1; p.N372H, p.H523R, p.I3412V, p.A2466V of BRCA2; p.RZ2P, p.R273C、p.R175H and p.C141Y of TP53. All the above mutations were proved to be SNPs except p.H523R of BRCA2and p.C141Y of TP53.Conclusions1. A total of34primary ovarian cancer xenograft models in nude mice were established. It provided a platform for further study. But the tumor biological behaviors such as growth pattern and metastasis ability were different from their originated tumors.2. Cisplatin, carboplatin and taisu could inhibit tumor growth within experimental period. It was similar with clinical initial treatment. The xenograft models in nude mice could be used to explore new anticancer drugs and to test drug susceptibility.3. Xenografts maintain the characteristics of human ovarian cancer gene mutation and morphological features.更多还原